Mechanism Of FSCN1 As RBP To Promote Bladder Cancer Invasion And Metastasis

Objective: According to the 2022 American cancer Society,Bladder cancer(BLCA)is the number one bladder cancer morbidity and mortality rate.BLCA can be divided into nonmuscle invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC).Surgery and chemotherapy are usually the first line of treatment for NMIBC,but many patients develop MIBC due to invasion and metastasis of BLCA cells.Due to the lack of accurate and effective targeted therapeutic drugs with few side effects,many patients with advanced bladder cancer and recurrent and metastatic bladder cancer can only continue their lives through chemotherapy or immunotherapy.Therefore,to clarify the invasion and metastasis mechanism of BLCA cells is the primary task to explore the therapeutic target of BLCA.As an actin binding protein,FSCN1 is usually associated with cellular pseudopodia formation,cell adhesion and motility.In breast cancer,liver cancer,melanoma,prostate cancer and BLCA,FSCN1 plays a role in promoting tumor cell invasion and migration.However,the mechanism by which FSCN1 promotes malignant progression of tumor cells in BLCA remains unclear.FSCN1 was first demonstrated as RBP in He La cells,and subsequently FSCN1 was believed to play the role of RBP in promoting the progression of nasopharyngeal carcinoma.RBP regulates RNA stability,transcription and translation.Therefore,we explored the mechanism by which FSCN1 as RBP plays a role in promoting tumor cell invasion and metastasis in BLCA.Methods:1.To detect the expression of FSCN1 in BLCA and its effect on tumor cell invasion and migrationAccording to the GEPIA website based on TCGA database,the expression level of FSCN1 mRNA in BLCA and the influence of FSCN1 as a risk factor on the prognosis of BLCA patients were obtained.The protein expression of FSCN1 in tumor tissues and paracancer tissues was detected by western blotting.Immunohistochemistry(IHC)assessed FSCN1 expression in tumor tissues and para-carcinoma tissues while FSCN1 expression in NMIBC and MIBC tissues was assessed.In BLCA cells,FSCN1 was silenced,and the expressions of E-cadherin,N-cadherin,and PD-L1 were detected by western blotting.After stable knockdown or overexpression of FSCN1 in BLCA cells,the invasion and migration ability of BLCA cells were detected by Transwell assay and scratch assay.2.To explore the mechanism of FSCN1 as RBP regulating TLN1 expression After RNA immunoprecipitation(RIP)sequencing in UM-UC-3 cells,it was found that FSCN1 could bind to the mRNA of TLN1.quantitative real-time PCR(qRT-PCR)and western blotting tests were used to detect the expression of TLN1 after FSCN1 was knocked down or overexpressed.The binding ability of FSCN1 and TLN1 mRNA was detected by RIP assay using FSCN1 antibody.RNA pulldown experiments were performed in BLCA cells or H293 T cells using RNA probes of TLN1.FISH kit was used to detect the location of TLN1 mRNA and FSCN1.After silencing FSCN1,statically knocking down FSCN1 and overexpressing FSCN1 in BLCA cells,the regulatory relationship between FSCN1 and TLN1 was detected by western blotting.3.The expression of TLN1 in BLCA and its effect on BLCA invasion and metastasis were detectedTLN1 expression in tumor tissues and paracancer tissues was detected by western blotting and IHC assay.The expressions of N-cadherin,β-catenin,P-GSK3β and GSK3β were detected after stable knocking of TLN1.Transwell assay and scratch assay were used to detect the invasion and migration ability of BLCA cells after TLN1 deletion.In vivo,6-week-old female BALB/c nude mice were used for lung metastasis model.T24 cells were used for stable knockdown of FSCN1 or TLN1,and the stable cells were injected into the tail vein of nude mice.After 3 weeks,PET-CT was performed on small animals to compare the number of tumors between the control group and the experimental group,and to judge the ability of BLCA cells to transfer after FSCN1 or TLN1 knockout.Results:1.FSCN1 promoted the invasion and migration of BLCA cells.The results of GEPIA database showed that the mRNA of FSCN1 in BLCA was highly expressed in tumor tissue,and survival analysis showed that the prognosis of patients with high FSCN1 expression was significantly worse.Subsequently,it was detected that the expression of FSCN1 protein in tumor tissue was higher than that in paracancer tissue.The expression of FSCN1 protein in MIBC tissue was higher than that in NMIBC group.Western blot assay showed that after silencing FSCN1,the contents of E-cadherin in BLCA cells increased,while the contents of N-cadherin and PD-L1 decreased.After FSCN1 knockdown,the invasion and migration ability of BLCA cells decreased,and after overexpression of FSCN1,the invasion and migration ability of BLCA cells were enhanced.2.FSCN1 binds to CDS region of RNA of TLN1 to promote the expression of TLN1 protein.RIP sequencing results showed that FSCN1 and TLN1 mRNA binding CDS region.After silencing or knocking down FSCN1,TLN1 protein expression decreased significantly.After overexpression of FSCN1,the protein expression of TLN1 was significantly increased.RIP and RNA pulldown experiments showed that the binding ability of FSCN1 and TLN1 mRNA in CDS region was stronger than that in 3’utr region in UM-UC-3 cells or HEK293 T cells.FISH and immunofluorescence experiments showed that mRNA colocalization of FSCN1 and TLN1 was detected.3.TLN1 promoted the invasion and migration of BLCA cells.Western blotting and IHC showed that TLN1 expression was higher in tumor tissue than in paracancer tissue.Transwell and scratch experiments showed that the migration and invasion ability of BLCA cells decreased after TLN1 knocking.Caudal vein models of nude mice showed that lung tumors were significantly reduced in the FSCN1 knockout and TLN1 knockout groups compared with the control group.Conclusions: FSCN1 increases its expression by binding to the CDS region of TLN1 mRNA.As RBP,FSCN1 contributes to the invasion and metastasis of BLCA cells.